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PubMed2004

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD.

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.

Catalysis↗
PubMed2002

Oxidation of sulfides and disulfides under electron transfer or singlet oxygen photosensitization using soluble or grafted sensitizers.

Two different photosensitizers, 9,10-dicyanoanthracene (DCA) and benzophenone (BzO) or a silica bound derivative (BzO-Si) have been compared for the photooxidation of di-n-butyl sulfide and di-n-butyl disulfide. With either photosensitizer, sulfide photooxidation in acetonitrile leads very efficiently to sulfoxide, with sulfone and disulfides as by-products. Although an electron transfer mechanism has previously been established starting with DCA, our results are indicative of two competitive mechanisms using BzO as the photosensitizer, instead of singlet oxygen addition and electron transfer. The more sluggish photooxidation of disulfides leads to a complex mixture of products, among which n-butyl butanethiosulfonate and strong acids (alkylsulfonic and sulfuric) are the major ones. The relative ratio thiosulfonate: acids depends, among other factors, on the medium polarity with acid formation favored starting with BzO or BzO-Si in a methanol-water mixture. An electron transfer mechanism only can account for the observed products Superoxide anion, the formation of which is much easier starting from BzO than from DCA, is suggested to play a crucial role in this oxidative radical pathway. Starting from disulfides, grafted benzophenone is more efficient for acid formation than its soluble counterpart. As this photosensitizer can easily be recycled, an easy and smooth way to acid formation is thus available, provided that the reaction solvent is properly chosen.

Anthracenes↗
PubMed2003

Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ.

We have developed a highly productive cell-free protein synthesis system from wheat germ, which is expected to become an important tool for postgenomic research. However, this system has not been optimized for the synthesis of disulfide-containing proteins. Thus, we searched here for translation conditions under which a model protein, a single-chain antibody variable fragment (scFv), could be synthesized into its active form. Before the start of translation, the reducing agent dithiothreitol, which normally is added to the wheat germ extract but which inhibits disulfide formation during translation, was removed by gel filtration. When the scFv mRNA was incubated with this dithiothreitol-deficient extract, more than half of the synthesized polypeptide was recovered in the soluble fraction. By addition of protein disulfide isomerase in the translation solution, the solubility of the product was further improved, and nearly half of the soluble polypeptides strongly bound to the antigen immobilized on an agarose support. This strong binding component had a high affinity as shown by surface-plasmon resonance analysis. These results show that the wheat germ cell-free system can produce a functional scFv with a simple change of the reaction ingredients. We also discuss protein folding in this system and suggest that the disulfide bridges are formed cotranslationally. Finally, we show that biotinylated scFv could be synthesized in similar fashion and immobilized on a solid surface to which streptavidin is bound. SPR measurements for detection of antigens were also possible with the use of this immobilized surface.

Amino Acid Sequence↗
PubMed2006

Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques.

The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds through the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur in mechanically stressed proteins. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by DTT. Although the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300-pN range. This result predicts that the disulfide bond lengthens by 0.34 A at the transition state of the thiol/disulfide exchange reaction. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property that has never been explored by traditional biochemistry. Furthermore, our work also indicates that the kinetics of any chemical reaction that results in bond lengthening will be force-dependent.

Connectin↗
PubMed2006

Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging.

Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii) occurs independently of endocytic vesicle trafficking along microtubules, and (iv) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.

Boron Compounds↗
PubMed2006

Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations.

The dissociable sigma subunit of bacterial RNA polymerase is required for the promoter-specific initiation of transcription. When bound to RNA polymerase, sigma makes sequence-specific promoter contacts and plays a crucial role in DNA melting. In isolation, however, sigma lacks significant promoter binding activity. In the crystal structure of the flagellar sigma factor, sigma(28), bound to the anti-sigma factor, FlgM, sigma(28) adopts a compact conformation in which the promoter binding surfaces are occluded by interdomain contacts. To test whether sigma(28) adopts this conformation in the absence of FlgM, we engineered a set of double cysteine mutants predicted to form interdomain disulfides in the conformation observed in the FlgM complex. We show that these disulfides form in both the presence and absence of FlgM. For two of the mutants, quantitative measurements of disulfide formation under equilibrium conditions suggest that the major solution conformation favors disulfide formation. The results indicate that the compact conformation of sigma(28) observed in the sigma(28)/FlgM structure is similar to the predominant conformation of free sigma(28) in solution. This finding suggests that autoinhibition of DNA binding in free sigma(28) is accomplished by steric occlusion of the promoter binding surfaces by interdomain interactions within the sigma factor as well as by a suboptimal distance between the promoter -10 and -35 element binding determinants in sigma(2) and sigma(4), respectively.

Amino Acid Substitution↗
PubMed2003

The role of disulfide bonds in the assembly and function of MD-2.

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDSPAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-kappaB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

Amino Acid Substitution↗
PubMed2003

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3.

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.

Amino Acid Sequence↗
PubMed1972

Covalent structure of bovine neurophysin-II: localization of the disulfide bonds.

The completed amino-acid sequence of bovine neurophysin-II, a major neurohypophyseal hormone-binding protein in the hypothalamo-neurohypophyseal complex of cows, set the stage for the localization of the disulfide bonds of this sulfur-rich molecule. Neurophysin-II was digested with subtilisin or a pepsin-trypsin mixture. The resulting peptides were subjected to first-dimensional electrophoresis at pH 6.5, oxidized with performic acid, and subjected to second-dimensional electrophoresis under identical conditions as the first-dimensional separation, but in a perpendicular direction. Cysteic acid peptides were eluted (several after additional electrophoretic purification at pH 3.5) for amino-acid composition and NH(2)- and COOH-terminal analyses. Our assignment of the seven disulfide bridges present in neurophysin-II is as follows: Cys(10)-Cys(93); Cys(13)-Cys(95); Cys(21)-Cys(27); Cys(28)-Cys(44); Cys(54)-Cys(61); Cys(67)-Cys(73); Cys(74)-Cys(79). The assignment of disulfide bridges associated with Cys(27) and Cys(28) is tentative as it is derived from evolutionary consideration. The high disulfide content reduces drastically the allowed number of biofunctional conformers of neurophysin-II. It is suggested that neurophysin-II possesses a globular topography with minimal alpha-helix structure.

Amino Acid Sequence↗
PubMed1975

Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain.

Tadpole collagenase (EC 3.4.24.3) cleaved chick cranial bone procollagen into two triple-stranded fragments, PCA and PCB. Only PCB, with an estimated molecular weight of about 60,000 for each component chain after reduction, was found to contain interchain disulfide bonds. The analogous cleavage of collagen is known to produce a large NH2-terminal fragment with a molecular weight of 70,000 for each chain and a small COOH-terminal fragment containing chains of about 25,000 molecular weight. Since PCB was too small to represent the product NH2-terminal to the site of collagenase cleavage, localization of interchain disulfide bonds to a COOH-terminal domain in procollagen was indicated. This assignment was conformed by Dintzis-type short-term labeling experiments. Procollagen obtained by acid extraction of bone lacked the COOH-terminal disulfide-bonded domain. The findings support a model for procollagen consisting of three proalpha chains each containing nonhelical NH2-terminal extensions of 20,000 molecular weight and COOH-terminal extensions of about 35,000 molecular weight, the latter linked by interchani disulfide bonds.

Animals↗
PubMed1978

The disulfide bond connecting the chains of ricin.

Studies on the disulfide bond connecting the two polypeptide chains of ricin are reported. Reduction of this bond in the native protein requires approximately 50-fold more mercaptoethanol than the reduction of the bond in the protein denatured by sodium dodecyl sulfate. An improved procedure for the formation of this disulfide bond from recombined chains is reported. A and B chains spontaneously and rapidly reassociate into a stable complex with a sedimentation velocity similar to that of native oxidized ricin before the disulfide bond reforms. The mixture of both chains also behaves on Bio-Gel P-100 like native oxidized ricin. However, the complex formed by the two chains, assayed before the disulfide bond can reform, and reduced ricin, carboxymethylated to prevent reoxidation, shows a significant decrease in toxicity to mice and a decrease in ability to inhibit protein synthesis in HeLa cells in culture.

Disulfides↗
PubMed1980

Differences in organizational structure of insulin receptor on rat adipocyte and liver plasma membranes: role of disulfide bonds.

Binding of 125I-labeled insulin to rat liver and adipocyte plasma membranes has been investigated after treatment of the membranes with agents that modify disulfide bonds or sulfhydryl groups. Dithiothreitol, a disulfide-reducing agent, produced a bimodal response in adipocyte plasma membranes with dose-dependent increases in binding occurring over the range of 0-1 mM dithiothreitol; 5 mM dithiothreitol produced decreased binding. Insulin binding reached its maximal increase at 1 mM and was 3 times control values. Scatchard analysis of the 1 mM dithiothreitol effect revealed a straight line plot indicative of one class of sites with a Ka of 1.0 x 10(8) M-1 which is intermediate between the two Kas obtained from the curvilinear Scatchard plot of control membranes. There was a 20-fold increase in the number of intermediate-affinity receptors compared to high-affinity receptors. The increased 125I-labeled insulin binding after dithiothreitol treatment was reversed by oxidized glutathione in a dose-dependent manner. Interposition of treatment with N-ethylmaleimide, an alkylating agent, prevented oxidized glutathione from reversing the dithiothreitol effect. Reduced glutathione produced the same effect as dithiothreitol. Liver plasma membranes treated with up to 1 mM dithiothreitol exhibited a maximum increase in insulin binding of 20% compared to control. Dithiothreitol at 5 mM decreased insulin binding below that of control membranes. The results indicate that the dithiothreitol effect on insulin binding to adipocyte plasma membranes is due to disruption of disulfide bonds, and that the structural organization of the insulin receptor on the plasma membranes is different for liver and for adipose tissue. The data imply that the insulin receptors on the plasma membrane of adipocytes possess at least two functionally distinct subclasses of disulfide bond but liver insulin receptors do not.

Adipose Tissue↗
PubMed1985

Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge.

The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfide bonds to transmit destructive energy. The radiation-dependent loss of A chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.

Disulfides↗
PubMed1985

Disulfide bond formation in the eye lens.

The disposition and disposal of the -SH groups of the lens during aging and cataractogenesis have been investigated by laser Raman spectroscopy as a noninvasive microprobe in the intact living lens. In this procedure -SH and -S-S- give unique discrete Raman signals (at 2580 and 508 cm-1) that may be used to calculate relative concentrations in a very small volume of the lens. We present evidence showing an unexpected and remarkable difference with respect to these groups between the mouse lens and the lenses of guinea pig and man. The mouse lens nucleus exhibits a precipitous fall in the -SH concentration on aging from 1 to 6 months; concomitantly, there is a rise in -S-S- of comparable magnitude, indicating a direct conversion. The guinea pig lens, however, is quite different with respect to the age-dependent change in nuclear -S-S-: there is none between 6 months and 5 years. In the human lens -S-S- behaves exactly as in the guinea pig lens: the level is low and does not change with age between 9 and 65 years. With respect to nuclear -SH, these two latter species of lenses show some decrease with age but nothing like the approach to zero found in the aging mouse lens nucleus. These differences involving lenticular -SH and -S-S- appear to be correlated with the hard nucleus in the mouse lens and the softer nuclei of lenses in guinea pigs and humans. The relatively high level of -S-S- in the old but clear mouse lens does not support the idea that protein aggregation involving formation of intermolecular -S-S- bonds is necessarily an important cause of nuclear cataract. The small but significant age-related depression of -SH in guinea pig lens nuclei without any accumulation of -S-S- may be explained as a result of glutathione (GSH) oxidation and subsequent extrusion of glutathione disulfide (GSSG) by the lens. We propose that the oxidation of glutathione proceeds by reaction with protein disulfide groups to yield protein sulfhydryl (PSH) and a mixed disulfide of glutathione and protein; the mixed disulfide is capable of being reduced by glutathione reductase and NADPH, yielding the original PSH and GSSG, which is extruded from the lens. It remains to be determined if this mechanism is more active in guinea pig and human lenses than in the mouse lens.

Aging↗
PubMed1986

Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen.

The third external domain of major histocompatibility class I antigens has a highly conserved disulfide bridge between cysteine-203 and cysteine-259. To elucidate the functional significance of this disulfide bridge, we have produced a mutant H-2Ld gene by site-directed mutagenesis in which the codon for cysteine-203 is changed to a codon for serine, which is unable to form a disulfide bridge. The mutant H-2Ld gene was introduced into mouse L cells and its expression has been studied. No measurable expression of the H-2Ld antigen was detected on the cell surface of the transformants by antibody-binding assays. However, a large quantity of the mutant H-2Ld antigen was found in the cytoplasm of the transformants as observed by immunoprecipitation of metabolically labeled lysate and by immunocytochemistry of membrane-permeabilized cells, using an antibody specific for the first external domain of the H-2Ld antigen. The mutant antigen was glycosylated and associated, at least in part, with beta 2-microglobulin. Subcellular fractionation experiments indicated that the transport of the antigen was blocked between the endoplasmic reticulum and the plasma membrane. It is concluded that structural integrity of the third external domain is a prerequisite for intracellular transport of class I antigens. On the basis of these findings we suggest that the domain structure containing the disulfide bridge serves as a signal structure necessary for receptor-mediated intracellular transport and that this requirement is the evolutionary basis for high conservation of similar structures present throughout the immunoglobulin supergene family.

Animals↗
PubMed1991

Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli.

The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS. These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function.

Amino Acid Sequence↗
PubMed1992

Determination of transmembrane protein structure by disulfide cross-linking: the Escherichia coli Tar receptor.

We have devised a generally applicable strategy for analysis of protein structure and have applied it to examine the structure of the transmembrane portion of the Tar receptor of Escherichia coli. The basis of our approach is the use of disulfide cross-linking to identify residues that are within close proximity. To generate and test large numbers of cysteine pairs, we used an unusual method of mutagenesis by which cysteine substitutions can be created randomly at a number of targeted codons. Cysteine-substituted proteins encoded by mutagenized genes may be screened directly for disulfide formation within oligomers or, alternatively, different pools of genes may be randomly recombined to generate gene populations with substitutions in multiple regions. Thus, it is possible to detect a variety of disulfide cross-links between and within individual protein molecules. Interactions between the four membrane-spanning stretches of the Tar dimer were probed by measuring the tendency of 48 cysteine substitutions throughout this region to form disulfide cross-links with one another. We have interpreted these data to suggest a helical-bundle structure for the transmembrane region. The four helices of this bundle are not structurally equivalent: the two TM1 helices interact closely, whereas the TM2 helices are more peripherally located.

Bacterial Proteins↗
PubMed1993

Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.

Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3 alpha (ubiquitin-protein ligase) and E2 (ubiquitin-carrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3 alpha.E2 complex are separated by approximately 20 A.

Amino Acid Sequence↗